It is known that certain facultative methanol utilizing yeasts, including Hansenula polymorpha, can be cultivated in an inorganic nutrient broth containing inorganic salts, vitamins and a carbon source and/or energy carrier which can be methanol.
In such systems, nutrient solution is continuously added, the cell mass is removed from the culture medium and the alcohol oxidase is recovered therefrom. Practically a steady state system can be provided in which the cell mass is removed at the same rate as the nutrient solution is added or vice versa. The cultivation of the yeast is effected at a pH of 4.0 to 6.0 and a temperature of 25.degree. to 45.degree. C. with the addition or air or/and air/oxygen mixture, the latter being oxygen-enriched air.
Alcohol oxidase is an enzyme which catalyzes the following reaction: ##STR1##
This enzyme can thus be used for the production of formaldehyde and/or for the production of hydrogen peroxide from methanol. It has also been used analytically for the determination of certain alcohols, generally methanol, ethanol, N-propanol and N-butanol with a measuring system utilizing a so-called enzyme electrode.
It is known in the art that methanol-consuming yeasts such as Candida, Hansenula, Pichia and Torulopsis stains, in cultivation or growth in the presence of methanol, induce a flavin-adenosine-dinucleotide (FAV) containing alcohol oxidase which catalyzes the oxidation of methanol to formaldehyde in a material exchange process.
It has been pointed out in the literature that for the recovery of alcohol oxidase enzyme, the yeast Candida boidinii and Hansenula polymorpha yeast can be used.
In the past Candida boidinii yeast has been used in batch cultures in a mineral-salt medium with methanol serving as a carbon and energy source with the vitamins biotine and thiamine being added.
These conventional processes are only limitedly effective. It has been pointed out that to achieve a higher cell density (higher density of the produced cell mass) additional methanol can be supplied during the growth or cultivation of the cells in the culture medium (see REUSS, M. et al, Chemie-Ingenieur-Technik, 46, pages 669 ff. (1974).
Upon termination of the yeast cultivation by this technique the yeast cells are found to have a specific activity of alcohol oxidase in the raw extract of 0.3 to 1.0 enzyme units per mg of protein.
In the recovery of alcohol oxidase from the methanol-consuming yeast Hansenula polymorpha, the culture medium is continuously supplied with a nutrient solution containing methanol and the vitamins biotine and thiamine. The quantity of alcohol oxidase obtained in this fashion, with the growth limited by the carbon and energy source methanol and with a growth rate of 0.6 h.sup.-1 (volume .times. volume .sup.-1 .times.h.sup.-1, i.e. volume rate of growth per total volume per hour), is 7% of the total protein, corresponding to a specific activity in the raw extract of 2.5 to 3.0 enzyme units per mg of protein.
With a growth rate of 0.03 h.sup.- under the same culture conditions, the alcohol oxidase quantity can be about 20% of the total protein so that the specific activity in the raw extract can correspond to 10 to 11 enzyme units per mg protein (see van DIJKEN, J.P.: Arch. Microbiol, 111, pgs. 137 ff, 1976).
After the termination of the cultivation, the enzyme is isolated from the yeast cells by column chromatography and enzyme precipitation.